Gene name - skittles
Cytological map position - 57B1
Function - kinase
Symbol - sktl
FlyBase ID: FBgn0016984
Genetic map position - 2-
Classification - 1-phosphatidylinositol-4-phosphate kinase
Cellular location - cytoplasmic
Skittles is a type of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) recently identified in Drosophila (Knirr, 1997a). Phosphoinositol lipids have been postulated to play important roles in various cellular processes including growth, differentiation, and vesicular secretion (for more information, see Drosophila Phosphotidylinositol 3 kinase 92E). The phosphatidylinositol pathway consists of a series of conversions of phosphatidylinositol (a membrane lipid bearing a sugar moiety attached to the lipid via an intermidiate phosphate residue) into singly, doubly, and triply phosphorylated products (Carpenter, 1996). An important branching point in the pathway occurs when phosphatidylinositol 4-phosphate (PtdInsP) is phosphorylated to become phosphatidylinositol 4,5-bis-phosphate (PtdIns[4,5]P2 or PIP2), a step catalyzed by phosphatidylinositol 4-phosphate 5-kinase (PIP5K; Boronenkov, 1995 and Ishihara, 1996). There are two types of PIP5Ks (PIP5KI and PIP5KII) with distinct biochemical and immunohistochemical properties, but they both catalyze the conversion of PtdInsP into PIP2 (reviewed in Loijens, 1996b). Skittles corresponds to a PIP5KI (Knirr, 1997a). The hydrolysis of PIP2 by phospholipase C (PLC) produces the second messengers diacylglycerol (DAG) and inositol tris-phosphate (IP3). DAG is an activator of protein kinase C (PKC: see Drosophila Protein kinase C), and IP3 plays an important role in the release of intracellular calcium. In addition, PIP2 is converted into phosphatidylinositol 3,4,5-tris-phosphate, which activates some PKC isoforms (Hassan, 1998 and references).
PIP2 is itself a second messenger that has been implicated in the modulation of the function of cytoskeletal regulatory proteins such as profilin, cofilin, fascin, and gelsolin (Janmey, 1994). There is also evidence that phosphoinositide metabolism is involved in signal transduction and cytoskeleton regulation via the interaction with the Rho family of small G proteins (Chong, 1994 and Ren, 1996). Rho and Rac small GTPases associate with type-I phosphatidylinositol 4-phosphate 5-kinase to regulate the production of phosphatidylinositol 4,5-bisphosphate. This lipid appears to mediate some of the effects of Rho and Rac on the actin cytoskeleton. The genes for several type-I phosphatidylinositol 4-phosphate 5-kinases have been cloned recently but it is not known which ones interact with Rho and/or Rac. Rho family GTPases also interact with phosphatidylinositol 3-kinase (see Drosophila Pi3K92E), though this kinase can be either upstream or downstream of the GTPases depending upon the system (Ren, 1998). Other work has suggested an interaction between phosphoinositides and receptor tyrosine kinases. It has also been suggested that PIP5K function may be associated with, or required for, DNA synthesis and cell proliferation. Finally, PIP5KI has been shown to be required for vesicular secretion in PC12 cells (Hay, 1995 ), while PIP5KII appears to be involved in vesicular trafficking in the budding yeast (Yamamoto, 1995). Most of the understanding of how PIP5Ks function to regulate cellular processes is derived from in vitro data. Whether the various, and apparently distinct, functions in which PIP5K is thought to be involved are related remains unknown. It also remains to be established whether these postulated roles for PIP2 are relevant in vivo and how the modulation of PIP5K levels affects development in animals (Hassan, 1998 and references).
The cloning of Drosophila skittles (sktl) makes the genetic and developmental analysis of the in vivo requirements of this gene possible and facilitates understanding the role(s) played by phosphoinositides in various tissues and cell types. sktl has been shown to be essential for cell and organism viability and is required for cytoskeletal regulation during sensory structure development. sktl is also required for germline development. This analysis resolves an issue pertinent to the function of another gene, inscuteable (insc). sktl maps to the first intron of insc, whose function is required for cell fate determination during neuronal and myogenic lineage development (Kraut, 1996a; Knirr, 1997b; Ruiz-Gomez, 1997 and Carmena, 1998). To date, all studies on neuronal insc function have been carried out using deletion alleles that remove or affect both genes, allowing for the possibility that the described phenotypes may be in part due to the loss of sktl or from the combined loss of sktl and insc. Hassan (1998) showed that the loss of sktl is not responsible for the insc phenotype.
Knirr (1997a) reported that sktl is expressed in germ cells during oogenesis. At stage 6, sktl expression is restricted to the future oocyte. By stage 9, expression is initiated in the nurse cells. At the end of oogenesis, large amounts of sktl transcript are present in the mature egg. To examine the function of sktl in germline development, sktl germline clones were generated using one of the excision alleles (sktlDelta15). Negative control crosses in which no recombination was induced result in 100% female sterility. The ovaries of these females, carrying the dominant sterile ovoD1 marker, are severely atrophic and show very early arrest of egg chamber development. Positive control crosses showed that 47% of the females were fertile. In contrast, all sktl recombinant females are sterile. Ovaries were dissected and stained with DAPI to reveal the nuclei. Oogenesis in these females is arrested after stage 10, and very few eggs are fully developed. Arrested egg chambers show defects in nurse cell nuclei at and after stage 10, but no defects in nuclear morphology are seen before that stage. The affected nuclei appear very small and fragmented, suggesting that sktl is required for nurse cell viability, and therefore proper egg chamber development. In contrast, the oocyte nucleus does not appear to be affected. The few eggs that did develop were smaller than the eggs produced by control flies and show defects in their dorsal appendages. Generally, the dorsal appendages of sktl mutant eggs are short and thick. The sterility associated with the partial loss of sktl in the female germline precludes the determination of the consequences of the loss of sktl in embryos (Hassan, 1998).
sktl is expressed widely in the wing disc. To examine the function of sktl in wing disc development sktl mutant clones were generated using the sktlDelta5 and sktlDelta15 alleles. The absence of the yellow marker was used to identify the clones. Approximately 76% of the control flies had yellow bristle clones on their notum, legs, and wing margin. These bristles showed no defects. In contrast, only 12% of the recombinant flies had yellow bristles on the notum. In addition the size of these clones was markedly reduced in comparison to control flies: each clone consisted of either a single bristle, or rarely, two bristles. The few mutant bristles recovered showed structural abnormalities ranging from a wavy shape (in most cases) to sharp bends (in a few cases), suggesting cytoskeletal defects. No mutant bristles were observed on the wing margin or the legs. The very small number of clones obtained combined with the small size of each clone suggest that during wing disc development sktl is required for either cell viability, proliferation, or both (Hassan, 1998).
To determine if sktl is required in the eye disc, where it is abundantly expressed, sktl mutant clones were generated. In control experiments, 45% of the recombinant flies had white eye clones of variable sizes. In contrast, no sktl mutant clones were observed, supporting the conclusion that sktl function is required for cell viability or cell division in imaginal discs. It should be noted that third instar larvae transheterozygous for the sktlDelta15 and famk07505 alleles show no defects in the sizes of the imaginal discs and the brain. Therefore it is more likely that the absence of sktl clones results from an effect on cell viability. While this hypothesis is favored, the possibility cannot be excluded that the sktlDelta15/famk07505 combination, while being lethal, is not severe enough to reveal a role for sktl in cell proliferation (Hassan, 1998).
The Drosophila adult peripheral nervous system has cells that extend sensory bristles rich in actin, providing an excellent system for the study of the defects in cytoskeleton assembly. Mutations in the Drosophila actin interacting proteins profilin (chickadee in Drosophila) and fascin (singed in Drosophila) show severe defects in bristle morphology. Loss of function of either protein results in bristles that lack actin filament integrity, causing bending and branching during extension. The analysis of Skittles indicates that the alteration of PIP5KI levels results in structural defects in sensory bristles, providing genetic evidence for the involvement of PIP5KIs in cytoskeletal regulation. Ectopic expression of Skittles causes ectopic bristle formation on the notum and wing blade. The appearance of ectopic bristles on the wing blade was seen in ~20% of the flies, but the majority of the flies (70%) showed ectopic bristles on the notum. These results prompted an examination of the effects of generalized overproduction of sktl in the wing disc. Using the 32B-Gal4 driver, which is expressed ubiquitously at high levels in the third instar wing disc, overexpression of sktl results in ectopic bristles (macrochaetae and microchaetae) on the notum and the wing blade. On the notum, both the ectopic and normal bristles showed severe structural defects. The structural defects of the bristles are consistent with a role for sktl in regulating cytoskeletal components. All ectopic bristles were associated with socket cells, suggesting that the production of ectopic bristles may be the result of the specification of extra sense organ precursors rather than the transformation of a socket cell into a bristle cell. The phenotypes observed in sktl clones are similar but not identical to those observed in singed and chickadee mutants. Specifically, while the sktl loss- and gain-of-function mutations resulted in bent and wavy bristles, no branching bristles were observed. Finally, it is interesting to note that mutations in all three genes (chickadee, singed, and sktl) result in female sterility. Therefore, both germline and bristle development present model systems in which to study the interactions between PIP5K and the actin cytoskeleton (Hassan, 1998).
The appearance of extra bristles associated with socket cells as a result of the overexpression of sktl in the wing disc can be explained in one of two ways: extra cell division, or specification of an extra precursor cell. The sterility resulting from the removal of the maternal component and the failure of somatic clones to survive does not allow for a correlation of the ectopic production of bristles with a loss-of-function phenotype. However, it is interesting to note that cytoskeleton-associated proteins like Inscuteable (Kraut, 1996a and Kraut, 1996b) and sanpodo (a Drosophila tropomodulin homologue; Dye, 1998) play a significant role in cell fate specification in the nervous system. This result suggests that sktl too may play a role in cell fate specification in the peripheral nervous system (Hassan, 1998).
Skittles bears a high degree of similarity to the human STM-7.1 gene. STM-7 has been assigned to the neurodegenerative disorder Friedrichs ataxia and codes for a PI4P5-kinase (Carvajal, 1996). Skittles shows similarity to a human placental PI4P5-kinase, two murine pancreatic PI4P5-kinases and two yeast genes: FAB1 and MSS1. Sktl shows 84% identity to the human STM-7 and the murine PI4P5K-1alpha protein and 60% identity to the yeast MSS4 protein (Knirr, 1997a).
date revised: 26 August 1999
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